Medical Devices and Coatings with Non-Leaching Antimicrobial Peptides

Abstract

Antimicrobial peptides enable an alternate approach to developing antimicrobial coatings due to their targeting of the membranes of the bacteria. High specific activity is achieved by orienting the peptides so that the antimicrobial ends of the peptides maximally contact the bacteria. In one embodiment, one end of the peptide is covalently attached directly to the substrate. In another embodiment, the peptides are immobilized on the substrate using a coupling agent or tether. Non-covalent methods include coating the peptide onto the substrate or physiochemically immobilizing the peptides on the substrate using highly specific interactions, such as the biotin/avidin or streptavidin system. The compositions are substantially non-leaching, antifouling, and non-hemolytic. The immobilized peptides retain sufficient flexibility and mobility to interact with and de endocytosed by the bacteria, viruses, and/or fungi upon exposure. Immobilizing the peptides to the substrate reduces concerns regarding toxicity of the peptides and the development of antimicrobial resistance, while presenting substantially all of the peptide at the site of action at the surface of the substrate.

1 . A composition comprising a substrate having immobilized thereon one or more antimicrobial peptides, wherein the antimicrobial peptides are uniformly tethered in a specified orientation.  
     
     
         2 . The composition of  claim 1  wherein the antimicrobial peptides are immobilized by bonds selected from the group consisting of covalent bonds, non-covalent bonds, and combinations of covalent and non-covalent bonds thereof.  
     
     
         3 . The composition of  claim 1  wherein the immobilized antimicrobial activity of the oriented peptides is greater than the immobilized antimicrobial activity of the same surface density and type of the peptides randomly tethered to the substrate without specified orientation.  
     
     
         4 . The composition of  claim 1  wherein the surface has immobilized antimicrobial activity at or below 0.2 mg/cm 2 , more preferably at or below 0.1 mg/cm 2 , even more preferably at or below 0.05 mg/cm 2 , and most preferably at or below 0.01 mg/cm 2 .  
     
     
         5 . The composition of  claim 1  wherein the peptides are oriented by specifically binding to their C-terminus.  
     
     
         6 . The composition of  claim 1  wherein the peptides are linear peptides.  
     
     
         7 . The composition of  claim 1  wherein the composition is substantially non-leaching and biocompatible.  
     
     
         8 . The composition of  claim 1  wherein the composition is substantially anti-fouling.  
     
     
         9 . The composition of  claim 1  wherein the composition is substantially non-cytotoxic.  
     
     
         10 . The composition of  claim 1  wherein the composition is substantially non-hemolytic.  
     
     
         11 . The composition of  claim 1  wherein the antimicrobial peptide sequence is between more than 9 and less than 150, more preferably less than 100, most preferably than 9-51, amino acids is length.  
     
     
         12 . The composition of  claim 1  wherein the antimicrobial peptide sequence is non-naturally occurring.  
     
     
         13 . The composition of  claim 1  wherein more than one peptide sequence is immobilized.  
     
     
         14 . The composition of  claim 1  wherein the immobilized antimicrobial activity of the oriented peptides is antibacterial.  
     
     
         15 . The composition of  claim 1  wherein the peptide is bound to the substrate by ionic binding.  
     
     
         16 . The composition of  claim 1  wherein the peptide is bound to the substrate by the interaction of strepavidin and biotin, polyhistidine-nickel chelate coupling, or salicylhydroxamic acid-phenyl boronic acid.  
     
     
         17 . The composition of  claim 1  wherein the surface of the substrate is modified through a gas-phase technique selected from the group consisting of plasma, corona discharge, flame treatment, UV/Ozone, UV and ozone only, aminolysis, hydrolysis, reduction, activation of alcohol chain ends with tosyl chloride and subsequent chemistry, graft copolymerisation of vinyl compounds by chemical initiation, or ion beam treatment in the presence of vinyl monomers.  
     
     
         18 . The composition of  claim 1  wherein the substrate surface is treated to introduce groups on the substrate surface, which can react with functional groups on the peptide, wherein the groups on the substrate are selected from the group consisting of hydroxyl, amine, halide, epoxide, activated ester, sulfhydryl, vinyl, and carboxylic acid groups.  
     
     
         19 . The composition of  claim 1  wherein thiol or amino groups in the peptides can react directly by conjugate addition reaction with unsaturated groups such as maleimides, vinyl sulfones, acrylamides and acrylates present in the substrate on the substrate.  
     
     
         20 . The composition of  claim 1  wherein the peptide is bound to the substrate by a functional group present in the peptide selected from the group consisting of amine, thiol, carbonyl, carboxyl, aldehyde, vinyl, phenyl, and alcohol.  
     
     
         21 . The composition of  claim 1  wherein one or more amine, alcohol or thiol groups on the peptide is reacted directly with a functional group on the surface of the substrate selected from the group consisting of isothiocyanate, acyl azide, N-hydroxysuccinimide ester, aldehyde, epoxide, anhydride, halides, sulphydryl, vinyl, and lactone.  
     
     
         22 . The composition of  claim 1  where one or more free amino, sulfhydryl or hydroxyl groups of the peptides are attached to a surface containing epoxide functional groups.  
     
     
         23 . The composition of  claim 1  comprising a tether or spacer molecule between the peptide and substrate.  
     
     
         24 . The composition of  claim 23  wherein the tether is hydrophilic polymer.  
     
     
         25 . The composition of  claim 24  wherein the tether is polyethylene glycol (PEG).  
     
     
         26 . The composition of  claim 23  wherein the peptide is coupled to the substrate with a homobifunctional sulfhydryl-reactive coupling agent.  
     
     
         27 . The composition of  claim 23  wherein the peptide is coupled to the substrate with a heterobifunctional sulfhydryl-reactive coupling agent.  
     
     
         28 . The composition of  claim 27  wherein the coupling agent is sulfo-GMBS.  
     
     
         29 . The composition of  claim 1  wherein a polymer is grafted onto the substrate and the peptides are covalently coupled to the polymer.  
     
     
         30 . The composition of  claim 29  wherein the polymer is crosslinked to form a gel.  
     
     
         31 . The composition of  claim 30  wherein the crosslinked polymer is Dextran.  
     
     
         32 . The composition of  claim 29  wherein the polymer is a polymer brush attached to the substrate.  
     
     
         33 . The composition of  claim 29  wherein the polymer is dendrimeric polymer attached to the substrate.  
     
     
         34 . The composition of  claim 29  wherein the polymer is synthesized by chemical vapor deposition.  
     
     
         35 . The composition of  claim 29  wherein the polymer is attached to a substrate formed of a material selected from the group consisting of silicone or polyurethane.  
     
     
         36 . The composition of  claim 1  wherein the peptide is attached to the substrate at a density of between 0.125 and 50 mg/cm 2 .  
     
     
         37 . The composition of  claim 1  wherein the peptide is attached to the substrate at a density of greater than 0.5 mg/cm 2 , more preferably 1 mg/cm 2 , even more preferably 5 mg/cm 2 , even more preferably 10 mg/cm 2 , and most preferably greater than 25 mg/cm 2 .  
     
     
         38 . The composition of  claim 1  wherein the antimicrobial activity remains for repeated uses with washing or storage for 21 days in organic or aqueous solvents between uses.  
     
     
         39 . The composition of  claim 1  wherein the substrate is a polymer, ceramic, or metal.  
     
     
         40 . The composition of  claim 39  wherein the substrate is in the form of an implantable or injectable device.  
     
     
         41 . The composition of  claim 40  wherein the device is selected from the group consisting of stents, catheters, tubing, needles, pacemakers, prosthetics, bone cement, screws, rivets, plates, valves, grafts, sensors, surgical instruments, and pumps.  
     
     
         42 . The composition of  claim 1  wherein the substrate is a tissue engineering or tissue culture support or matrix.  
     
     
         43 . The composition of  claim 1  wherein the substrate is fibrous.  
     
     
         44 . The composition of  claim 43  wherein the fibrous substrate is in the form of a device selected from the group consisting of gauze, pads, wound dressings, surgical drapes, surgical garments, diapers, and sponges.  
     
     
         45 . The composition of  claim 1  wherein the substrate is a membrane.  
     
     
         46 . The composition of  claim 1  wherein the substrate is in the form of nanoparticles, microparticles or beads.  
     
     
         47 . The composition of  claim 1  wherein the substrate further comprises one or more therapeutic, prophylactic, or diagnostic agents which are covalently tethered to the surface or optionally released independently of the immobilized antimicrobial peptide.  
     
     
         48 . The composition of  claim 47  wherein the therapeutic, prophylactic or diagnostic agent is selected from the group consisting of antiproliferative, cytostatic, or cytotoxic chemotherapeutic agents, antimicrobial agents, anti-inflammatory agents, growth factors, antithromobotic agents, and cell adhesion peptides.  
     
     
         49 . The composition of  claim 47  wherein the therapeutic, prophylactic or diagnostic agent is tethered to the substrate using a hydrolyzable linkage so that the agent is slowly released from the substrate.